Ground glass hepatocytes (GGHs), a histological hallmark of chronic hepatitis B virus (HBV) infection, contain excessive hepatitis surface antigen (HBsAg) in the endoplasmic reticulum (ER), which is linked to unfolded protein response (UPR). The mechanism by which HBV activates UPR has not been fully defined. To investigate this, HepG2-NTCP cells and primary human hepatocyte (PHH) were either infected with HBV or transduced with adenoviral vectors expressing replication-competent HBV genome or individual HBV genes. UPR markers were evaluated by qPCR, Western blotting, and immunofluorescence. Apoptosis and cell viability were measured by Caspase3/7 and ATPlite assay respectively. We found that UPR markers were induced by the overexpression of HBsAg in HepG2-NTCP cells and PHHs. Elevation of UPR-induced genes showed a dose-dependent correlation with HBsAg levels. In HBV-infected livers, GGHs also demonstrated excessive accumulation of HBsAg associated with increased BIP/GRP78 staining, a marker of UPR. Prolonged activation of UPR by HBsAg overexpression induced signs of apoptosis. Overexpression of HBsAg can induce ER stress through protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway in vitro, and may be linked to the appearance of GGHs. The activation of UPR by HBsAg may sensitize hepatocytes to cell death and result in possible subsequent cellular changes leading to a premalignant phenotype. HBV remains a major public health problem with about 300 million people chronically infected worldwide. HBV has a compact genome which only codes for 4 major gene families, and the virus depends heavily on host factors for infection and replication. Comprehensive knowledge of critical host dependencies could provide valuable insight into the molecular mechanisms of viral replication and facilitate the development of novel host targeting agents. To systematically identify host factors involved in HBV infection, we used a HepG2 cell line that overexpressed NTCP, a recently identified host receptor for HBV. This cell line was then optimized and miniaturized for a high-throughput AlphaLisa screen to detect viral antigen. An arrayed genome-wide screen targeting approximately 21,500 genes using three unique siRNA sequences per gene was performed using this HepG2-NTCP cell line and AlphaLisa assay to detect viral antigen post-infection. A counter-screen with a cell viability assay measuring ATP as an endpoint assay was performed to assess the effect of knockdown on cytotoxicity. The candidate genes found included known or previously identified host factors, such as SLC10A1, HNF4A, CH25H, RXRA, PLK1 etc, and also many additional host cell proteins that were not known previously to be involved in HBV infection. The identification and characterization of host factors participating in the complete HBV lifecycle will both advance our understanding of HBV pathogenesis and uncover novel therapeutic targets.